Water soluble defructosylated pea extract, and use thereof as a prebiotic agent

ABSTRACT

The present invention relates to a water-soluble defructosylated pea extract, substantially free of proteins and peptides, as well as to an oligosaccharide composition such as that contained in said extract. It also relates to the method for preparing the same, and to the use thereof as a prebiotic agent.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a National Stage of International Application No.PCT/FR2010/000238 filed Mar. 22, 2010, claiming priority based on Frenchpatent application Ser. No. 09/01511 filed Mar. 27, 2009, the contentsof all of which are incorporated herein by reference in their entirety.

The present invention relates to a water-soluble defructosylated peaextract, substantially free of proteins and peptides, as well as to anoligosaccharide composition such as that contained in said extract. Theinvention also relates to the method for preparing the same, and to theuse thereof as a prebiotic agent.

Legumes constitute a choice raw material for the agro-processingindustry, particularly for the production of proteins, starch,particularly amylose-rich starch, fibers and starch derivatives such asglucose syrups, maltodextrin, dextrose or isoglucose.

These products find outlets in varied fields, such as the adhesive orpaper sectors, and especially in the food sector whereby the nutritionalvalue of the legumes, for human consumption as much as for animalfeeding, no longer has to be proven. Among the legumes, grain legumessuch as beans, peas, and fava beans, are widely used for their energyand protein content. The grains of dried peas are indeed rich incarbohydrates, formed essentially of starch as well as of saccharose andoligosaccharides, in proteins (with a high lysine content) and infibers.

In exchange for their nutritional benefits, the legumes such as driedpeas have a low digestibility, which often requires soaking them in anacidic medium before they can be cooked and eaten. This drawback ismainly due to their significant content of α-galactosyl oligosaccharidesconstituted of units of D-galactose, D-glucose and D-fructose. Indeed,these oligosaccharides, which cannot be digested by human enzymes(incapable of degrading their α-1,6-galactosidic and β-1-4 fructosidicbonds), are transported intact up to the colon where they provide asubstrate for the fermentation of bacteria such as Clostridiumperfringens, causing flatulence. This phenomenon, according to someauthors, in the case of beans (Phaseolus vulgaris), has been attributed,in particular, to the fructose terminal motif of the raffinose whichthey contain (MYHARA R M et al., Can. Inst. Food Sci. Technol. J., Vol.21, no 3, pp. 245-250, 1988).

These oligosaccharides are thus generally eliminated either by way ofagronomic lineage selection (particularly soybean or bean) low in [ . .. ] such as oligosaccharides (BURBANO C. et al., J. Sci. Food Agric.,Vol. 79, pp. 1468-1472, 1999), or by separation and physicalelimination, or else by enzymatic hydrolysis (by means of anα-galactosidase) or fermentative hydrolysis, carried out, in general,before the consumption of these legumes, but also by taking foodsupplements constituted of enzymes adapted to hydrolyze theseoligosaccharides into digestible compounds, before they reach the colon(U.S. Pat. No. 5,651,967).

U.S. Pat. No. 4,008,334 thus proposes a method for eliminating thesoluble carbohydrates from the vegetable proteins originating, inparticular, from soybean, including raffinose and stachyose, by enzymedigesting by means of baker's yeast. Similarly, U.S. Pat. No. 4,216,235suggests using a Saccharomyces uvarum yeast to degrade theoligosaccharides of soybeans, including melibiose and manninotriose.

In the case of the pea, the oligosaccharides are most often eliminatedduring the commercial transformation of the legume grains. It isestimated that, currently, the volume of oligosaccharides generated bythe refining of peas is experiencing a strong growth and is greater than25,000 metric tons per year in Europe and in Canada. To avoid having toeliminate them in a purification plant, and thus, in spite of their lowdigestibility, the latter are generally handed off to farmers to be usedas livestock feed. However, this solution has a high energy cost since,because of the substantial dilution of the soluble extract originatingfrom the refining, it is necessary to concentrate it to ensure itspreservation and enable its transportation and its use by animals.

It would thus be interesting to be able to have a means that would makeit possible to valorize these by-products.

With that in mind, document US 2004/0198965 suggested to useoligosaccharides, present, in particular, in soybean seeds, for thesynthesis of D-galactose.

The invention relates to another use of these by-products. Indeed, theApplicant has discovered that a pea soluble fraction, substantially freeof proteins and peptides and rich in oligosaccharides, could, afterdefructosylation of the oligosaccharides, be usefully used as prebioticagent.

The term “prebiotic” is to be understood as a non-digestible,selectively fermented food component which causes specific changes tothe composition and/or activity of the intestinal microbiota, beneficialto the health and well-being of the host (Gibson G R et al., NutritionResearch Reviews, 17: 259-275, 2004). The prebiotic can in particular beconsidered as an aliment for favorable bacteria of the colon, such asthe bifidobacteria and the lactobacilli, which enable preventingintestinal disorders, enhancing the absorption of minerals, modulatingthe lipid metabolism, and/or stimulating the immune system.

Since it has been discovered that it was possible to modulate thebalance of the intestinal microbiota by ingesting food ingredients,numerous candidates to the appellation “prebiotic” have been studied.Most are carbohydrates of vegetable origin. The most known and thebetter characterized are the fructans, fructose polymers, among whichare the inulin, generally extracted from chicory tubers, but also fromagave, and the fructo-oligosaccharides (FOS) produced either byhydrolysis of inulin, or by biosynthesis from saccharose and fructose.

Other oligosaccharides also have prebiotic properties that are more orless established: the galacto-oligosaccharides (GOS) or“trans-galacto-oligosaccharides” (TOS), very close to the compoundspresent in mother's milk, the soy oligosaccharides (SOS), theisomalto-oligosaccharides (IMOS), the lactulose, the raffinose, thexylo-oligosaccharides, etc.

In spite of the numerous studies carried out on the already wellcharacterizes prebiotics (such as the FOS and the GOS) as well as on agreat number of candidate molecules, there is still, to this day, a lackof a prebiotic combining the following properties which are sought afterby the (potential) producers of food and drinks with a healthfunctionality: a simple and economical production method, very goodintestinal tolerance allowing for the use of adequate concentrations toobtain, in practice, the desired results, and high stability to heatingprocesses and to the conventional acidic media of large sectors of thefood industry.

Thus, the inulins and FOS have an average intestinal tolerance.According to certain authors, symptoms of discomfort with the FOS ariseas low as 2.5 g/day for 25% of the subjects, and affect 75% of thesubjects at 20 g/d. The tolerance to inulins is comparable to that ofthe FOS, but their effective dosage to obtain a bifidogenic effect ishigher, which intensifies the arbitration problem between a minimumdosage to obtain a bifidogenic effect and a maximum dosage to preventthe problems of intestinal tolerance. Furthermore, the inulins and theFOS are not stable to acids and heat.

This is not the case for the XOS, the raffinose, and the SOS which, inaddition, provide good prebiotic activity. However, the latter have avery low intestinal tolerance.

Pyrodextrins, IMOS, and polydextrose, which present a good stability toacids and heat and a good tolerance, have, on the other hand, a lowefficiency because of a low prebiotic specificity (their stimulation ofcolic bacteria is undifferentiated) and/or low indigestibility.

For their part, the GOS have an intestinal tolerance and a highstability to acidity and to processes. They are obtained by reaction oftrans-galactosylation (polymerization) from lactose by means ofβ-galactosidases. The reaction yield is low, negatively impacted by thesynthesis of the desired compounds (β-GOS), which means that varioustechnologies for separation, particularly membrane or chromatographic,are required on the reaction product to make it possible to maintainconditions that are favorable to the trans-galactosylation and toachieve a product that is sufficiently pure for prebiotic use. Thesetechnologies are costly and thus counter to affordability of GOSprebiotics.

This results in the existing prebiotics being used only in a limitednumber of food applications (humanized milk in particular) and atconcentrations which do not always make it possible to guarantee theeffects theoretically possible according in vitro/vivo, or even clinicalresearch.

However, the Applicant has demonstrated that oligosaccharide mixturespresent in defructosylated pea extracts make it possible to achieve thedesired compromise of properties for prebiotics.

The object of the present invention is thus a water-soluble pea extractsubstantially free of proteins and peptides and defructosylated.

It also has as an object a composition having the oligosaccharideprofile of these water-soluble defructosylated pea extracts and whichholds, and preferably is mostly constituted of, melibiose,manninotriose, and mannino-tetraose with a weight ratiomannino-tetraose/melibiose of at least 1:1 and preferably of at least4:1 or even of at least 5:1 (and a maximum of about 10:1, for example)and/or a weight ratio manninotriose/mannino-tetraose of 0.3:1 to 4:1,preferably of 0.8:1 to 1:1.

It also has as an object the use as prebiotic agent of either awater-soluble defructosylated pea extract substantially free of proteinsand peptides and defructosylated, or the aforementioned composition.

What is meant by “substantially free of proteins and peptides” is thatthe water-soluble extract according to the invention holds less than 5%in weight (dry matter) of such constituents. The latter include, inparticular, the albumines.

The defructolysated pea extract according to the invention can beobtained by defructosylating the oligosaccharides present in thesolubles of peas substantially free of proteins and peptides.

The method for obtaining this extract can in particular comprise thefollowing steps:

-   -   1—Separation of the flocculatable proteins, insoluble fibers        and/or starch of the pea or pea flour to obtain a water-soluble        pea extract,    -   2—Elimination of the soluble peptides, in particular of the        antitrypsic factors,    -   3—Action of an invertase, originating, for example, from        Saccharomyces cerevisiae, on said extract, under conditions        allowing for the defructosylation of the oligosaccharides        present in said extract, and    -   4—Recovery of the defructosylated extract thus obtained.

The invention thus also has as an object a method for preparing awater-soluble defructosylated pea extract substantially free of proteinsand peptides comprising at least the successive steps describedhereinabove.

It must be understood that this method can, as explained below, compriseother preliminary, intermediary or subsequent steps to those indicatedhereinabove. Thus, it can also include at least one demineralization(desalting) steps to reduce the reactions of coloration in certainapplications. Before the other minerals are eliminated, a step ofelimination of the potash (by crystallization, for example) similar tothat carried out on sugar vinasse, is advantageously carried out. Thesalts present in the pea solubles being particularly rich in potassium(on the order of 35%), this treatment has the dual advantage of reducingby about 50% the mineral content left to be eliminated by othertechniques (particularly ion-exchange resins) and of generating somepotash which constitutes a highly-valued fertilizer, allowed in organicagriculture. This treatment generally consists of an acidification(often with H2SO4) followed by a neutralization with ammonia, thecrystals being recovered by decanting and/or centrifugation.Alternatively, membrane techniques (particularly electrodialysis) thatmake it possible to optimize the method while reducing costs can beused.

Such method is generally implemented on a native pea. The pea accordingto the invention can be a variety of smooth or wrinkled peas and isadvantageously chosen from among those of the species Pisum sativum L.,particularly among the subspecies: elatius, transcaucasicum Govorov,syriacum Berger, abyssinicum Govorov, asiaticum Govorov, sativum(varieties Arvense L., sativum or macrocarpon, for example).

The peas can be first washed, sorted, trimmed and/or dusted off beforethey are grinded into a flour which is then suspended in water.

The separation of the flocculatable proteins, starch and/or insolublefibers can be done by any means known to one having ordinary skill inthe art, and particularly by extraction, centrifugation, decanting,membrane filtration (particularly ultrafiltration) or isoelectricprecipitation of the proteins or by a combination of several of thesemeans, possibly combined with a concentration step, for example byreverse osmosis and/or vacuum evaporation. An example of such separationmethod is described in application WO 2007/017572.

The pea solubles obtained are rich in peptides and in oligosaccharides.These pea solubles are processed to separate the peptides, which have amolecular weight greater than 5,000 Da, advantageously byultrafiltration and/or nanofiltration. It must be noted that therecoverable peptide fraction is enriched with albumin PA1b (having amolecular weight of 11,000 Da) whose insecticide properties suitable tocereals are described in application EP 1 078 085. The recovery of thefraction for such non-food use thus constitutes an additional potentialadvantage of the present invention.

At the end of this step, a water-soluble fraction rich inoligosaccharides and substantially free of proteins and peptides andwhich can advantageously by concentrated by reverse osmosis is obtained.

The defructosylation of the oligosaccharides of residual peas containedin this fraction can thus be carried out in a continuous or sequentialmanner, by any method of acid, thermal and/or enzyme hydrolysis allowingfor eliminating the terminal fructose motif from the oligosaccharidespresent in the pea solubles, while preserving their α-galactosidicbonds, and in particular while subjecting them to the action of anenzyme or of yeast (advantageously Saccharomyces cerevisiae) having aninvertase (or β-fructofuranosidase) activity. This step makes itpossible to transform respectively the raffinose, the stachyose, and theverbascose into melibiose, manninotriose, and mannino-tetraose.

The free enzyme (invertase of Saccharomyces cerevisiae) can, forexample, be implemented in a quantity of 200 to 300 UI.mg⁻¹ dry, at atemperature of 40 to 50° C., advantageously with a pH of 5.0 to 5.4, fora duration comprised, for example, between 10 to 14 h, on anultrafiltrate concentrated at 100-400 μl, for example at about 200 g/l,in a proportion of 50 to 70 UI of enzyme per gram of dry matter of theultrafiltrate.

The use of yeast rather than an enzyme in this defructosylation methodpresents the advantage that the yeast uses, and thus eliminates, thefructose produced by the reaction, which makes it possible to recoverdirectly (without later separation) a product without sugar. In the casewhere the method according to the invention uses an enzyme, it shallcomprise, on the contrary, an additional step for eliminating thefructose.

This method presents several non-negligible environmental advantageswith respect to the methods for obtaining prebiotics of the prior art,insofar as:

-   -   The raw materials that are used are preferably waste products of        the agro-processing industry,    -   It advantageously calls upon membrane purification techniques,        which consume little energy,    -   It makes it possible to have recourse to natural biotechnologies        by using non-modified conventional microorganisms.

The soluble defructolysated pea extract obtained can be used in liquidform or possibly in powder form after having been dehydrated,particularly by atomization or freeze-drying. Its use in liquid form ispreferred since, due to its low average molecular weight and to itspolydispersity, the product provides a low viscosity, does not causecrystallization problems and provides good microbiological stability.

It has been demonstrated that the oligosaccharide profile of thedefructosylated pea is particularly advantageous in terms of the soughtafter prebiotic efficiency and of its tolerance with respect tooligosaccharide mixtures originating from other defructosylated legumes,such as soybeans or beans. In particular, the laxative effect of the peais less than that of the soybean and it has prebiotic effects not onlyat the entrance of the colon, but also in the proximal and median colon.Furthermore, the defructosylation method is less complex and costly inthe case of the pea than with other legumes such as soybeans or beans,insofar as it does not require a step of separating the sucrose or ofdemineralization, which, on the other hand, is indispensable in the caseof soybeans, whose solubles are very rich in mineral salts.

In addition, because of the elimination of the fructose, thedefructosylated pea extract according to the invention is hypocariogenicand has a low caloric value.

The defructosylated pea extract according to the invention and thesimilar compositions thus constitute a prebiotic ingredient of choice inview of at least one of the following benefits being obtained:modification of the bacterial composition of the colon, stimulation ofthe bifidobacteria and lactobacilli, diminution of the relative quantityof harmful bacteria and pathogenic germs such as Clostridium, E. coli,diminution of the luminal pH, increase of mineral absorption, inparticular of calcium, stimulation of detoxifying bacterial enzymes,repression of toxifying bacterial enzymes, insolubilization of the bileslats enhancing their excretion, inhibition of the biliary acidconversion, diminution of the production of harmful compounds such asphenols and indoles, diminution of constipation, decreased risk ofinfectious diarrheas, decrease of inflammatory bowel diseases,stimulation of the production of short-chain fatty acids, stimulation ofthe apoptosis, prevention of colon cancer, reinforcement of theintestinal barrier, diminution of the bacterial translocation and/or ofbacterial toxins, stimulation of the secretion of satiation hormones,diminution of the cholesterol level, weight reduction, diminution ofweight gain, diminution of food intake, adipogenesis reduction and/orreduction of the symptoms of metabolic irregularity.

They are thus advantageously used to these ends, or for the fabricationof a composition adapted to obtain at least one of the aforementionedbenefits.

In addition, because it is easy to obtain and inexpensive tomanufacture, the defructosylated pea extract according to the inventionis well-suited for use in consumer market products. It is particularlywell-adapted for use in shelf-stable products and sweet carbonateddrinks because of its stability in storage, heat, and acids.

In these applications, the defructosylation of the pea extract can becarried out before its incorporation with a food product or drink towhich it is intended to provide prebiotic properties. Alternatively, thedefructosylation can be carried out during the preparation treatmentand/or during storage (under the action of the acidity or invertaseactivity present in such food product or in such drink).

The present invention thus has as an object a carbonated drink having atleast one sweetener and one water-soluble defructosylated pea extractand/or a composition such as previously described.

It also has for an object a food such as a shelf-stable food, a yogurtor baby milk containing a water-soluble defructosylated pea extractand/or a composition such as previously described.

Alternatively, the defructosylated pea extract according to theinvention, or the corresponding composition, can be administered as anutritional supplement, for example in the form of syrup, capsule,tablet, powder for oral suspension, or any other galenic form adapted tooral administration.

According to an embodiment of the invention, the drink, the food, or thenutritional supplement containing the defructosylated pea extractaccording to the invention, or the corresponding composition, can alsoinclude at least one other prebiotic agent, particularly originatingfrom defructosylated soybean solubles, defructosylated fava bean, ordefructosylated beetroot vinasses.

The invention will now be illustrated by way of the non-limitingexamples that follow.

EXAMPLES Example 1 Preparation of a Water-Soluble Defructosylated PeaExtract

A pea water-soluble fraction, at 50% weight of dry matter obtained afterextraction of the starch and fibers and coagulation of the proteins, isdiluted at 15% weight of dry matter and filtered by means of anultrafiltration membrane, with a cutoff fixed at 5,000 Da, so as toclarify it and remove the peptides from it. This step is followed by aconcentration of the ultrafiltrate by reverse osmosis, to bring it backto 20% weight of dry matter.

At the same time, 100 ml of a solution of invertase at 1 mg/ml isprepared and subsequently washed by centrifugation for 30 minutes. Thepellet is collected from 50 ml of water. An amount of 980 ml of the peafraction is then mixed with 50 ml of the enzyme solution in adouble-wall reactor with agitator placed in a water bath at 50° C. Thehydrolysis is controlled by dosage of the reducing sugars by means of anaqueous alkaline solution of 3.5-dinitrosalicylic acid (DNS), atdifferent time intervals. After at least 12 h of hydrolysis, the enzymeis neutralized, then the product obtained is centrifuged then filteredto obtain a clear solution which is then concentrated by vacuum rotaryevaporation at 70° C. until a clear juice is obtained.

Example 2 Study of Digestive Tolerance of the Water-SolubleDefructosylated Pea Extract

2-1: Objective of the Study

The objective of this study was to compare the digestive tolerance ofthe product originating from the present invention (defructolysatedoligosaccharides: DO) to that of two prebiotics already on the marketand to that of a placebo.

This study of digestive tolerance has been carried out in France on 17volunteers (8 men and 9 women), double-blind.

2-2: Test Procedure

The volunteers having participated in this study have consumed, in arandom manner, 20 g of active substance diluted in orange juice in onedose, each dose being spaced apart by a 48-hour rest period, to achievefour doses, total.

The different products tested were:

-   -   defructosylated oligosaccharides (DO: product of the invention),    -   β-galacto-oligosaccharides (β-GOS),    -   fructo-oligosaccharides (FOS),    -   saccharose (placebo)

The digestive tolerance was evaluated by means of a questionnairereporting on the frequency (graded from 1: no symptom to 5: veryfrequent symptoms) and the intensity (graded from 1: no symptom felt to5: substantial and/or unacceptable symptoms) from 6 differentparameters:

-   -   abdominal pain,    -   bloating,    -   abdominal noises (borborygmi, gurgling sounds . . . ),    -   flatulence,    -   nausea, regurgitation or heartburn,    -   need to defecate        2-3: Results and Conclusions of the Study

The results of the study are presented in Table 1 herein below, whichregroups the average grades obtained for each parameter subsequent tothe evaluation of digestive tolerance of DOs, β-GOS, and FOS compared tothat of a placebo.

TABLE 1 Abdominal pain Bloating Abdominal noises Flatulence Nausea Needto defecate Cumulative F I F I F I F I F I F I grade placebo 1.1^(a)1.1^(a) 1.2^(a) 1.2^(a) 1.2^(a) 1.2^(a) 1.9^(a) 1.5^(a) 1.0^(a) 1.0^(a)1.6^(a) 1.2^(a) 15.1^(a) DO 1.4^(a) 1.4^(a) 1.9^(b) 1.5^(a,b) 2.0^(b)1.8^(b) 3.1^(b) 2.6^(b) 1.2^(a) 1.2^(a) 2.0^(a) 1.8^(a) 21.9^(b) β-GOS1.6^(a,b) 1.5^(a,b) 1.6^(a,b) 1.7^(b) 1.8^(b) 1.6^(a,b) 2.9^(b) 2.5^(b)1.2^(a) 1.1^(a) 2.1^(a) 1.7^(a) 21.3^(b) FOS 2.0^(b) 1.9^(b) 2.1^(b)1.9^(b) 2.1^(b) 2.0^(b) 3.2^(b) 2.7^(b) 1.0^(a) 1.0^(a) 2.1^(a) 2.1^(a)24.2^(b) F: frequency of the symptoms; I: intensity of the symptoms.^(a,b)values of the products having a different letter are significantlydifferent from the other values (p < 0.05; Duncan's differentiationtest)

It emerges from Table 1 that the frequency and severity of the symptomsobserved from the consumption of the different products tested haveshown that the latter did not cause digestive intolerance (cumulativegrades about equal to the third of the maximum grade).

However, certain differences between products have been observed:

-   -   the DOs were as well tolerated as the β-GOS (product having the        best tolerance among the prebiotics currently on the market)        compared to the placebo (which has the lowest grades for all the        symptoms)    -   the DOs have shown better digestive tolerance than the FOS        (which remains the product that is the least well-tolerated with        the highest grades for all the symptoms).

This study thus demonstrates the satisfactory digestive tolerance of theproduct originating from the invention.

Example 3 Study of the Bifidogenic Effect of the Water-SolubleDefructosylated Pea Extract

To evaluate the bifidogenic effect of the extract according to theinvention on the intestinal microbiota, a randomized, monocentric,double-blind, placebo-controlled study was carried on 36 healthyvolunteers (men and women from 20 to 45-years old) distributed into twoequal groups, as outpatients. The product according to the invention wasconsumed at a dose of 7 g of oligosaccharides (representing 95% drymatter) per day in one dose, whereas the placebo was constituted ofsyrup made of dehydrated glucose consumed at the same dosage. Theduration of the treatment was 3 weeks. The bifidobacteria were dosed inreal-time by quantitative PCR.

Example 4 Study of Stability in Acidic Medium of the Water-SolubleDefructosylated Pea Extract

The stability in acidic medium of the defructosylated pea extractaccording to the invention was evaluated on sugar-free cola-type drinkshaving a pH of 2.8. The tests were carried out in comparison with anidentical but not defructosylated pea extract.

To this end, half of the bottles of each of these lots, containing 2.48g of the tested extract, was stored at ambient temperature and the otherhalf was stored at 37° C., for one month.

Samples of each lot kept at each temperature were collected twice aweek. The samples were analyzed by HPLC in order to determine thevariation of their concentration in oligosaccharides over time, andthus, the stability of the latter.

The invention claimed is:
 1. A composition consisting essentially ofmelibiose, manninotriose, and mannino-tetraose, having a weight ratiomannino-tetraose/melibiose of at least 1:1 or a weight ratiomanninotriose/mannino-tetraose of 0.3:1 to 4:1.
 2. The compositionaccording to claim 1, wherein the weight ratio ofmannino-tetraose/melibiose is least 4:1.
 3. The composition according toclaim 2, wherein the weight ratio mannino-tetraose/melibiose is at least5:1.
 4. The composition according to claim 1, wherein weight ratio ofmanninotriose/mannino-tetraose is 0.8:1 to 1:1.
 5. The compositionaccording to claim 1, wherein the weight ratio ofmannino-tetraose/melibiose is at least 5:1 and the weight ratio ofmanninotriose/mannino-tetraose is 0.8:1 to 1:1.